Construction And Immune Protective Efficacy Of Recombinant Adenovirus Expressing Hemagglutinin Gene Of H5N1Avian Influenza Virus

Highly pathogenic avian influenza （HPAI） is a highly contagious and fatal disease hazarded tothe poultry industry. Since1997, H5N1viruses have been spreading throughout the world, andhave broke through the species barrier to infect a variety of mammals, including human. Currently,vaccination is still the most effective way to combat avian influenza pandemic. The traditionalwhole virus inactivated vaccine depended on the chick embryo production can not meet thedemand. Therefore, seeking new avian influenza vaccine with easy production, low costs, andefficient is of important significance for people to prevent and control avian influenza.Adenovirus vectors as a delivery vehicle of the therapeutic gene have advantage of wide hostrange, good safety, simple immune pathway and efficient expression of exogenous genes. So it hasa broad application prospects.In order to construct recombinant adenovirus expressing clade2.3.4and clade2.3.2.1influenza virus HA gene, HA gene of A/wild duck/Shandong/628/2011（H5N1）（SD628）and A/wildduck/Shan dong/2/2011（H5N1）（SD2） were amplified by RT-PCR and inserted into adenovirusshuttle plasmid pShuttle-CMV to construct recombinant adenovirus shuttle plasmid pShuttle-CMV-SD628-HA、pShuttle-CMV-SD2-HA. rAd-HA （rAd-SD628-HA and rAd-SD2-HA）were generatedby the Ad-Easy technique and produced the cytopathic effect in293cells. The adenovirus particleswere observed through cell ultramicrotome. Further more, the transcription and expression of HAgene in Ad293cells were also vertified by RT-PCR, IFA and western blot. The result showed that ithas a similar immunoreactivity as influenza viral protein. Generation P4recombinant adenoviruswas purified by adenovirus purification kit. The virus titers of rAd-SD628-HA, rAd-SD2-HA were10¹⁰TCID₅₀/mL,7.5×10⁹TCID₅₀/mL respectively.BALB/c mice were vaccinated with the rAd-SD628-HA by intramuscular injection andintranasal immunization respectively. The immunization doses of both groups were10⁸TCID₅₀,5x10⁸TCID₅₀respectively. Three weeks after the primary vaccination, mice were boostervaccinated with the same way. The serum antibody levels were tested by neutralization test （VN）.One week after booster immunization, VN antibodies of Intramuscular immune group, intranasalimmune group reached to1:113,1:66respectively. The antibody titer presented a significantupward tend. After challenged with the homologous virus SD628, the mice were in good condition,no death, and its weight appeared smaller fluctuations in adenovirus immune group; but the weight of control mice reduced with19%on the average, and two mice died. It was demonstrated thatrAd-SD628-HA was capable of inducing immune responses to protect mice against lethal dosesH5N1influenza viruses through both of in intramuscular and intranasal immunization.SPF chickens were vaccinated with the rAd-SD628-HA by intramuscular injection. Threeweeks after the primary vaccination, booster immunzation. Two weeks after booster immunization,chickens were challenged with the homologous virus SD628and the heterologous virus SD2,which belonged to Clade2.3.4, Clade2.3.2.1respectively. The serum antibody levels were tested byhemagglutination inhibition test （HI）. When detected by antigen A/wild duck/Shandong/628/2011,HI antibodies of10⁸TCID₅₀dose group,10⁹TCID₅₀dose group, Re-6inactivated vaccine groupwere9log2,9.16log2and6.88log2after booster immunization. After challenged with thehomologous virus SD628, the protective efficiency was87.5%, and three chickens shed virus in10⁸TCID₅₀dose group. Compared with them,100%protection was provided by10⁹TCID₅₀rAd-SD628-HA and Re-6inactivated vaccine, and no chicken, three chickens shed virusrespectively. However, all chickens of the control group were dead in six days. The results showedthat the immune protection and inhibition of shed virus provided by10⁹TCID₅₀dose were betterthan10⁸TCID₅₀dose against homology H5N1virus, and the protective efficacy was equivalent tothe inactivated vaccine.In the other hand, when detected by antigen A/wild duck/Shandong/2/2011（H5N1）, two weeksafter booster immunization, HI antibodies of10⁹TCID₅₀dose group, Re-6vaccination group were4log2,10.88log2respectively. Two weeks after challenged with the heterologous virus SD2, HIantibodies reached to6.14log2,11.14log2, respectively. Both groups provided100%protection forchickens. Four chickens shed virus in10⁹TCID₅₀dose group and no chicken shed virus in Re-6vaccination group. However, all chickens were dead in three days in control groups. The resultsshowed that10⁹TCID₅₀rAd-SD628-HA can protect SPF chickens against heterology H5N1viruslethal attacks.In this study, the H5N1influenza recombinant adenovirus live vector vaccine was able toelicit a strong antibody response against lethal H5N1AIV in BALB/c mice and SPF chickens.Those provide a theoretical basis for the research and development of new influenza vaccines.